Polypeptides having glucoamylase activity

ABSTRACT

Polypeptides having the amino acid sequence represented by SEQ ID NO:6 in Sequence Listing or an amino acid sequence derived from the above sequence by at least one of deletion, addition, insertion or substitution of one or more amino acids and showing a thermophilic glucoamylase activity.

TECHNICAL FIELD

The present invention relates to a polypeptide, more specifically, apolypeptide having a glucoamylase activity, which is useful forprofitable utilization of biomass. The present invention also relates toa gene that is useful for producing said polypeptide by geneticengineering.

BACKGROUND ART

Two enzymes each having an activity of releasing D-glucose linked at anon-reducing end through an α-1,4 bond, glucan 1,4-α-glucosidase andα-glucosidase, are known.

Glucan 1,4-α-glucosidase (EC 3.2.1.3) is also called 1,4-α-D-glucanglucohydrolase or glucoamylase. It is an enzyme-that acts on anon-reducing end of a polymer of D-glucopyranose linked through α-1,4bonds to release β-D-glucose. Glucan 1,4-α-glucosidases derived from thefollowing are currently known: fungi such as those of genus Aspergillus,genus Mucor, genus Rhizopus, genus Piricularia, genus Thermomyces andgenus Trichoderma; yeasts such as those of genus Endomyces and genusSaccharomyces; and a bacterium of genus Clostridium. This enzyme, likeα-amylase, is an important enzyme used in a process of hydrolyzingstarch. Thus, the enzyme is industrially utilized in wide variety offields including production of glucose, isomerized sugars andoligosaccharides, as well as production of liquors and fermentedalcohol.

Glucose is usually produced from starch as follows. Starch isgelatinized by cooking. The gelatinized starch is liquefied by allowingα-amylase to act on it at about 80 C. Then, saccharification is carriedout by allowing glucan 1,4-α-glucosidase to act at 55 to 60 C. Theliquefaction is carried out at a high temperature because thegelatinized starch is highly viscous and because thermostable α-amylaseshave been put to practical use. It is desirable to select a temperatureof 55 C. or above for the saccharification in order to avoidcontamination with microorganisms. However, if a commonly used enzymederived from a fungus is used, one must select a temperature of 60 C. orbelow because of the thermostability of the enzyme. Accordingly, it isimpossible to carry out the steps of liquefaction and saccharificationat the same time because their optimal temperatures are different fromeach other, resulting in a great wasteful cost for energy.

α-Glucosidase (EC 3.2.1.20) is an enzyme that acts on an α-glucosidebond at a non-reducing end to release α-D-glucose. It is widely presentin animals, plants and microorganisms. α-Glucosidases are classifiedinto groups (1) to (3) based on the substrate specificity as follows:(1) ones that act on a wide variety of hetero and homo α-glucosidecompounds; (2) ones that are highly specific forα-1,4-glucooligosaccharides and that have relatively weak activities onhigh molecular weight glucan and heteroglucoside; and (3) ones that arehighly specific for α-1,4-glucoside bonds or that also act on starch orglycogen. Among these, those belonging to the group (3) are often calledglucoamylases (“Oyo Kosogaku”, Tsujisaka et al. (eds), Kodansha (1979)pp. 56).

Hyperthermophilic microorganisms, which are adapted to high temperatureenvironment, produce highly thermostable enzymes. Pyrococcus furiosus, ahyperthermophilic archaebacterium, is known to producesaccharide-hydrolyzing enzymes such as α-amylase, α-glucosidase,β-glycosidase and β-glucanase. Genes for some of these enzymes have beencloned (The Journal of Biological Chemistry, 268:24402-24407 (1993); TheJournal of Biological Chemistry, 272:16335-16342 (1997); Journal ofBacteriology, 172:3654-3660 (1990); Journal of Bacteriology,177:7105-7111 (1995); Journal of Bacteriology, 181:284-290 (1999)).However, there is no known hyperthermophilic microorganism producingglucan 1,4-α-glucosidase or hyperthermostable glucan 1,4-α-glucosidase.Furthermore, known α-glucosidases produced by hyperthermophilicmicroorganisms including Pyrococcus furiosus act only on low molecularweight substrates and do not digest high molecular weight substratessuch as starch.

OBJECTS OF INVENTION

The main object of the present invention is to provide an industriallyadvantageous polypeptide having a thermostable glucoamylase activity, agene encoding said polypeptide and a method of producing saidpolypeptide by genetic engineering.

SUMMARY OF INVENTION

The present invention is outlined as follows. The first aspect of thepresent invention relates to a polypeptide having the amino acidsequence of SEQ ID NO:6 or an amino acid sequence in which one or moreamino acid residue is deleted, added, inserted and/or substituted in theamino acid sequence of SEQ ID NO:6 and having a thermostableglucoamylase activity. The second aspect of the present inventionrelates to a nucleic acid encoding the polypeptide of the first aspect.The third aspect of the present invention relates to a nucleic acidencoding a polypeptide having a thermostable glucoamylase activity whichis capable of hybridizing to the nucleic acid of the second aspect understringent conditions. The fourth aspect of the present invention relatesto a recombinant DNA containing the nucleic acid of the second or thirdaspect. The fifth aspect of the present invention relates to atransformant transformed with the recombinant DNA of the fourth aspect.The sixth aspect of the present invention relates to a method forproducing the polypeptide of the first aspect, the method comprisingculturing the transformant of the fifth aspect and collecting apolypeptide having a thermostable glucoamylase activity from theculture. The seventh aspect of the present invention relates to a methodfor producing glucose, the method comprising allowing the polypeptide ofthe first aspect to act on a polymer of D-glucopyranose liked throughα-1,4 bonds to release glucose. The eighth aspect of the presentinvention relates to a method for producing an oligosaccharide, themethod comprising allowing the polypeptide of the first aspect to act ona polymer of D-glucopyranose liked through α-1,4 bonds to generate anoligosaccharide. The ninth aspect of the present invention relates to amethod for producing a cyclodextrin, the method comprising allowing thepolypeptide of the first aspect to act on a polymer of D-glucopyranoseliked through α-1,4 bonds to generate a cyclodextrin.

As a result of intensive studies, the present inventors have found thata gene encoding a novel polypeptide exists on the Pyrococcus furiosusgenome. Furthermore, the present inventors have surprisingly found thatthe polypeptide has a thermostable glucoamylase activity although theamino acid sequence of the polypeptide is highly homologous only tothose of various α-amylases and cyclomaltodextrin glucanotransferasesderived from bacteria. The present inventors have further investigatedto establish a method for producing the polypeptide by geneticengineering. Thus, the present invention has been completed.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the relationship between the reaction temperature andthe glucoamylase activity of the polypeptide of the present invention.

FIG. 2 illustrates the relationship between the reaction pH and theglucoamylase activity of the polypeptide of the present invention.

FIG. 3 illustrates the remaining glucoamylase activity of thepolypeptide of the present invention upon heating at 80° C.

FIG. 4 illustrates the remaining glucoamylase activity of thepolypeptide of the present invention upon heating at 95° C.

FIG. 5 illustrates the relationship between the concentration of a metalion or EDTA and the glucoamylase activity of the polypeptide of thepresent invention.

FIG. 6 illustrates the composition of products obtained by allowingrespective crude extracts to act on maltopentaose.

FIG. 7 illustrates the composition of products obtained by allowingrespective crude extracts to act on soluble starch.

DETAILED DESCRIPTION OF THE INVENTION

1. The Polypeptide of the Present Invention

The polypeptide of the present invention has the amino acid sequence ofSEQ ID NO:6 or an amino acid sequence in which one or more amino acidresidue is deleted, added, inserted and/or substituted in the amino acidsequence of SEQ ID NO:6, and has a thermostable glucoamylase activity.

As used herein, a glucoamylase activity means an activity thatsequentially hydrolyzes glucoside bonds in a polysaccharide or anoligosaccharide consisting of D-glucopyranose linked through α-1,4 bondsfrom the non-reducing end to release D-glucose. An enzyme that releasesβ-D-glucose is called glucan 1,4-α-glucosidase (EC 3.2.1.3). An enzymethat releases α-D-glucose is called α-glucosidase (EC 3.2.1.20). As usedherein, having a glucoamylase activity means that at least one of thetwo catalytic activities is exhibited.

Methods for determining a glucoamylase activity are exemplified by knownmethods. Such methods include a method in which an enzymatic reaction iscarried out using amylose as a substrate, and the increase in the amountof reducing sugar is determined according to the Park & Johnson method,and a method in which D-glucose released upon the same enzymaticreaction is measured according to an enzymatic method using glucoseoxidase.

The polypeptide of the present invention has a glucoamylase activity.The polypeptides of the present invention also include polypeptides thathave, in addition to the glucoamylase activity, other activities such asa hydrolase activity (e.g., an α-amylase activity or a β-amylaseactivity) or a glycosyltransferase activity (e.g., a cyclomaltodextringlucanotransferase). For example, the polypeptide of the presentinvention having an amino acid sequence of SEQ ID NO:6 has aglycosyltransferase activity because when an oligosaccharide such asmaltose, maltotriose or maltotetraose is used as a substrate, itgenerates glucose and a product having a higher molecular weight thanthat of an oligosaccharide as a substrate.

The polypeptide of the present invention has a thermostable glucoamylaseactivity. Although it is not intended to limit the present invention,“having a thermostable glucoamylase activity” means that the polypeptideexhibits a glucoamylase activity at a temperature of 70° C. or above,preferably 80° C. or above, more preferably 85° C. or above, mostpreferably 90° C. or above.

The polypeptides of the present invention include a polypeptide havingan amino acid sequence in which one or more amino acid residue isdeleted, added, inserted and/or substituted in the amino acid sequenceof SEQ ID NO:6 as long as it has a thermostable glucoamylase activity.

A mutation such as deletion, insertion, addition or substitution of anamino acid in an amino acid sequence may be generated in a naturallyoccurring polypeptide. Such mutation may be generated due to apolymorphism or a mutation of the DNA encoding the polypeptide, or dueto a modification of the polypeptide in vivo or during purificationafter synthesis. However, it is known that such a mutated polypeptidemay exhibit physiological or biological activities substantiallyequivalent to those of a polypeptide without a mutation if such amutation is present in a portion that is not important for the retentionof the activity or the structure of the polypeptide.

This is applicable to a polypeptide in which such a mutation isartificially introduced into an amino acid sequence of a polypeptide. Inthis case, it is possible to generate more various mutations. Forexample, it is known that a polypeptide in which a cysteine residue inthe amino acid sequence of human interleukin-2 (IL-2) is replaced by aserine retains the interleukin-2 activity (Science, 224:1431 (1984)).

Furthermore, it is known that certain polypeptides have peptide regionsthat are not indispensable to their activities. Such peptide regions areexemplified by a signal peptide in a polypeptide to be secretedextracellularly or a prosequence found in a precursor of a protease.Most of such regions are removed after translation or upon conversioninto an active polypeptide. Such a polypeptide has a primary structuredifferent from that of a polypeptide without the region to be removed,but finally exhibits an equivalent function. A gene having a nucleotidesequence of SEQ ID NO:2 which is isolated according to the presentinvention encodes a polypeptide having the amino acid sequence of SEQ IDNO:1. This polypeptide has a thermostable glucoamylase activity. Asignal peptide-like sequence consisting of 19 amino acid residues ispresent at the amino terminus of the encoded polypeptide. A polypeptidein which the signal peptide has been removed from the polypeptide, i.e.,the polypeptide having the amino acid sequence of SEQ ID NO:6, also hasa thermostable clucoamylase activity. Thus, the polypeptides of thepresent invention include both of the above-mentioned two polypeptides.

When a polypeptide is produced by genetic engineering, a peptide chainthat is irrelevant to the activity of the polypeptide of interest may beadded at the amino terminus or the carboxyl terminus of the polypeptide.For example, a fusion polypeptide, in which a portion of an aminoterminus region of a polypeptide that is expressed at a high level inthe host to be used is added at the amino terminus of the polypeptide ofinterest, may be expressed in order to increase the expression level ofthe polypeptide of interest. In another case, a peptide having anaffinity with a specific substance may be added at the amino terminus orthe carboxyl terminus of the polypeptide of interest in order tofacilitate the purification of the expressed polypeptide. The addedpeptide may remain added if it does not have a harmful influence on theactivity of the polypeptide of interest. If necessary, it may beengineered such that it can be removed from the polypeptide of interestby appropriate treatment, for example, by limited digestion with aprotease.

Thus, a polypeptide having an amino acid sequence in which one or moreamino acid residue is deleted, inserted, added and/or substituted in theamino acid sequence disclosed herein (SEQ ID NO:1) is encompassed by thepresent invention if it has a thermostable glucoamylase activity.

The polypeptides of the present invention include the mutantpolypeptides as described in Examples below such as F206S, P142I, L337V,FS/PI and FS/LV.

The polypeptide of the present invention can be produced, for example,by (1) purification from a culture of a microorganism producing thepolypeptide of the present invention, or (2) purification from a cultureof a transformant containing a nucleic acid encoding the polypeptide ofthe present invention.

(1) Purification from culture a of a microorganism producing thepolypeptide of the present invention

The microorganism producing the polypeptide of the present invention isexemplified by Pyrococcus furiosus DSM3638 which can be purchased fromDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. Themicroorganism is cultured under conditions suitable for the growth ofthe microorganism. Preferably, culture conditions that increase theexpression level of the polypeptide of interest are used. Thepolypeptide of interest produced in the cells or the culture medium canbe purified according to a method conventionally used for purifying aprotein.

A method conventionally used for culturing a hyperthermophile can beutilized for the cultivation of the above-mentioned strain. Nutrientsthat can be utilized by the strain are added to the culture medium. Forexample, starch can be used as a carbon source, and Tryptone, peptoneand yeast extract can be used as nitrogen sources. A metal salt such asa magnesium salt, a sodium salt or an iron salt may be added to aculture medium as a trace element. In addition, it may be advantageousto use artificial seawater for the preparation of a culture medium, forexample. A clear culture medium that does not contain solid sulfur isdesirable. By using such a culture medium, the growth of cells can bereadily monitored by measuring the turbidity of the culture.

The culture may be a standing culture or a spinner culture. For example,a dialysis culture method as described in Applied and EnvironmentalMicrobiology, 55:2086-2088 (1992) may be used. In general, the culturetemperature is preferably about 95° C. Usually, a considerable amount ofa polypeptide is accumulated in the culture after culturing for about 16hours. It is preferable to determine the culture conditions depending onthe strain or the composition of the culture medium to be used such thatthe productivity of the polypeptide becomes maximal.

A cell-free extract is first prepared in order to obtain a polypeptide.The cell-free extract can be prepared, for example, by collecting cellsfrom a culture by centrifugation, filtration or the like and thendisrupting the cells. A cell disruption method highly effective forextracting the enzyme of interest may be selected from sonication,disruption using beads, treatment with a lytic enzyme and the like. Ifthe polypeptide is secreted into a culture supernatant, the polypeptidein the culture supernatant is concentrated by ammonium sulfateprecipitation, ultrafiltration or the like. The concentrated polypeptideis used as a cell-free extract. A method conventionally used forpurifying a protein can be used to isolate the polypeptide from the thusobtained cell-free extract. For example, ammonium sulfate precipitation,ion exchange chromatography, hydrophobic chromatography, gel filtrationchromatography and the like can be used in combination.

(2) Purification from Culture of Transformant Transformed withRecombinant DNA Containing Nucleic Acid Encoding the Polypeptide of thePresent Invention

The polypeptide of the present invention can be obtained from atransformant transformed with a recombinant DNA that contains a nucleicacid encoding the polypeptide of the present invention, for example, anucleic acid having a nucleotide sequence of SEQ ID NO:2 or 7. Apolypeptide having an amino acid sequence of SEQ ID NO:1 is producedusing a nucleic acid having a nucleotide sequence of SEQ ID NO:2. Apolypeptide having an amino acid sequence of SEQ ID NO:6 is producedusing a nucleic acid having a nucleotide sequence of SEQ ID NO:7.

The host to be transformed is not limited to specific one andexemplified by those conventionally used in a field of recombinant DNAincluding Escherichia coli, Bacillus subtilis, yeast, filamentous fungi,plants, animals, plant cultured cells and animal cultured cells.

For example, the polypeptide of the present invention can be obtainedusing Escherichia coli JM109 harboring pSJ3231, a plasmid in which a DNAhaving a nucleotide sequence of SEQ ID NO:2 is linked downstream from alac promoter. Escherichia coli JM109 transformed with pSJ3231 isdesignated and indicated as Escherichia coli JM109/pSJ3231, anddeposited on Jul. 30, 1999 (the date of the original deposit) underBudapest Treaty at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan under accession number FERM BP-7196.

The polypeptide can be expressed in cultured cells by culturingEscherichia coli JM109 harboring pSJ3231 under conventional cultureconditions, for example, in LB medium (10 g/l Tryptone, 5 g/l yeastextract, 5 g/l NaCl, pH 7.2) containing 100 μg/ml of ampicillin at 37°C. until logarithmic growth phase, addingisopropyl-β-D-thiogalactopyranoside at a final concentration of 0.2 mMthereto and further culturing at 37° C.

Cells are collected by centrifugation after cultivation, disrupted bysonication, and a supernatant collected by centrifugation is used as acell-free extract. This cell-free extract exhibits a thermostableglucoamylase activity. The polypeptide of the present invention can bepurified from the cell-free extract by using known methods such as ionexchange chromatography, gel filtration, hydrophobic chromatography andammonium sulfate precipitation. Naturally, a partially purified productobtained during the purification process as described above alsoexhibits a glucoamylase activity. Since the polypeptide of the presentinvention expressed in Escherichia coli JM109 harboring pSJ3231 ishighly thermostable, the cultured cells and/or the cell-free extract maybe heated, for example, at 80° C. for 10 minutes to removeheat-denatured insoluble proteins derived from the host in order topurify the polypeptide.

Alternatively, the polypeptide of the present invention having the aminoacid sequence of SEQ ID NO:6 can be obtained in a similar manner usingEscherichia coli harboring pET21amyCΔS as described in Examples below.

As described above, when the polypeptide of the present invention isexpressed at normal temperature (e.g., 37° C.) using a transformantharboring a nucleic acid encoding the polypeptide, the resultingexpression product retains the activity, the thermostability and thelike. That is, the polypeptide of the present invention can assume itsinherent higher-order structure even if it is expressed at a temperaturequite different from the growth temperature of the original producercell.

Some of the enzymological properties of the polypeptide of the presentinvention obtained as described above (e.g., the polypeptide having anamino acid sequence of SEQ ID NO:6) are shown below.

(1) Action:

The polypeptide of the present invention hydrolyzes amylose to generateglucose and oligosaccharides.

In addition, the polypeptide of the present invention acts on amylose togenerate a cyclodextrin.

(2) Optimal Temperature: it Exhibits Maximum Activity at 85-90 C.

(3) Optimal pH: it Exhibits Maximum Activity at pH 5-6.

(4) The Stability is Increased in the Presence of CaCl₂.

(5) The Activity is Inhibited by EDTA.

2. The Nucleic Acid of the Present Invention

The nucleic acid of the present invention is a nucleic acid that encodesthe polypeptide of the present invention as described above.Specifically, it is exemplified by (1) a nucleic acid encoding apolypeptide having the amino acid sequence of SEQ ID NO:6 or an aminoacid sequence in which one or more amino acid residue is deleted, added,inserted and/or substituted in the amino acid sequence of SEQ ID NO:6and having a thermostable glucoamylase activity; (2) a nucleic acidhaving the nucleotide sequence of SEQ ID NO:7; and (3) a nucleic acidencoding a polypeptide having a thermostable glucoamylase activity whichis capable of hybridizing to the nucleic acid of (1) or (2) above understringent conditions.

As used herein, a nucleic acid means a single-stranded ordouble-stranded DNA or RNA. If the nucleic acid of (2) above is an RNA,it is represented by a nucleotide sequence in which T is replaced by Uin the nucleotide sequence of SEQ ID NO:7, for example.

For example, the nucleic acid of the present invention can be obtainedas follows.

The nucleic acid of (2) above having the nucleotide sequence of SEQ IDNO:7 can be isolated as follows. A genomic DNA is obtained according toa conventional method from Pyrococcus furiosus DSM3638 cultured asdescribed above for the polypeptide of the present invention. Thegenomic DNA is used to construct a DNA library. The nucleic acid can beisolated from the DNA library. Also, the nucleic acid can be obtained byamplifying a nucleic acid having a nucleotide sequence of SEQ ID NO:7 bya polymerase chain reaction (PCR) using the genomic DNA as a template.

Furthermore, a nucleic acid encoding a polypeptide having a thermostableglucoamylase activity similar to that of the polypeptide of the presentinvention can be obtained on the basis of the nucleotide sequence of thenucleic acid encoding the polypeptide of the present invention providedby the present invention (e.g., the nucleotide sequence of SEQ ID NO:7).Specifically, a DNA encoding a polypeptide having a thermostableglucoamylase activity can be screened by using the nucleic acid encodingthe polypeptide of the present invention or a portion of the nucleotidesequence as a probe for hybridization from a DNA extracted from cells orPCR products obtained using the DNA as a template. Alternatively, a DNAencoding a polypeptide having a thermostable glucoamylase activity canbe amplified using a gene amplification method such as a PCR using aprimer designed based on the above-mentioned nucleotide sequence.Additionally, a DNA encoding a polypeptide having a thermostableglucoamylase activity can be chemically synthesized. The nucleic acidsof (1) or (3) above can be obtained according to such a method.

A nucleic acid fragment containing only a portion of the nucleic acid ofinterest may be obtained according to the above-mentioned method. Inthis case, the entire nucleic acid of interest can be obtained asfollows. The nucleotide sequence of the obtained nucleic acid fragmentis determined to confirm that the fragment is a portion of the nucleicacid of interest. Hybridization is carried out using the nucleic acidfragment or a portion thereof as a probe. Alternatively, a PCR iscarried out using a primer synthesized on the basis of the nucleotidesequence of the nucleic acid fragment.

“Hybridize under stringent conditions” refers to being capable ofhybridizing under conditions as described in T. Maniatis et al. (eds.),Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring HarborLaboratory (1989), for example, under the following conditions. Amembrane onto which a nucleic acid is immobilized is incubated with aprobe in 6×SSC (1×SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0)containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1%polyvinylpyrrolidone, 0.1% Ficoll 400 and 0.01% denatured salmon spermnucleic acid at 50° C. for 12 to 20 hours. After incubation, themembrane is washed in 2×SSC containing 0.5% SDS at 37° C. while changingthe SSC concentration down to 0.1× and the temperature up to 50° C.until the signal from the immobilized nucleic acid can be distinguishedfrom background, and the probe is then detected. The activity of theprotein encoded by the thus obtained novel nucleic acid is determined asdescribed above, thereby confirming whether or not the nucleic acid isthe nucleic acid of interest.

If an oligonucleotide probe is used, “stringent conditions” refer to,for example, incubation at a temperature of [Tm−25° C.] overnight in asolution containing 6×SSC, 0.5% SDS, 5×Denhardt's and 0.01% denaturedsalmon sperm nucleic acid although it is not intended to limit thepresent invention.

Tm of an oligonucleotide probe or primer can be determined, for example,according to the following equation:Tm=81.5−16.6(log₁₀[Na+])+0.41(% G+C)−(600/N)wherein N is the chain length of the oligonucleotide probe or primer; %G+C is the content of guanine and cytosine residues in theoligonucleotide probe or primer.

If the chain length of the oligonucleotide probe or primer is shorterthan 18 bases, Tm can be estimated, for example, as the sum of theproduct of the number of A+T (adenine and thymine) residues multipliedby 2(° C.) and the product of the number of G+C residues multiplied by4(° C.): [(A+T)×2+(G+C)×4].

According to the present invention, a nucleic acid which is capable ofhybridizing to the nucleic acid encoding the polypeptide of the presentinvention under stringent conditions is encompassed by the presentinvention as long as it encodes a polypeptide having a thermostableglucoamylase activity even if it does not have the same nucleotidesequence as that disclosed herein, as described above.

It is known that one to six codon(s) (a combination of three bases),which defines an amino acid in a gene, is assigned for each amino acid.Thus, many nucleic acids can encode one specific amino acid sequencealthough it depends on the amino acid sequence. Nucleic acids are notnecessarily stable in nature. Generation of a mutation in a nucleotidesequence is not unusual. A mutation generated in a nucleic acid may notalter the encoded amino acid sequence (called a silent mutation). Inthis case, it can be said that a different nucleic acid encoding thesame amino acid sequence is generated. Thus, it cannot be denied thatvarious nucleic acids encoding the same amino acid sequence can begenerated in the course of passage of an organism containing a nucleicacid encoding one specific amino acid sequence. Furthermore, it is notdifficult to artificially produce various nucleic acids encoding thesame amino acid sequence if one uses various genetic engineeringtechniques.

For example, if a codon used in an original nucleic acid encoding aprotein of interest is one whose codon usage is low in the host to beused for producing the protein by genetic engineering, the expressionlevel of the protein may be low. In this case, the codon is artificiallyconverted into one frequently used in the host without altering theencoded amino acid sequence aiming at elevating the expression level ofthe protein of interest (e.g., JP-B 7-102146). As described above,various nucleic acids encoding one specific amino acid sequence can beartificially prepared, of course. They may also be generated in nature.

The nucleic acid encoding the polypeptide of the present invention(e.g., a nucleic acid having the nucleotide sequence of SEQ ID NO:7) canbe ligated to an appropriate vector to construct a recombinant DNA. Thevector to be used for the construction of the recombinant DNA is notspecifically limited. For example, plasmid vectors, phage vectors andvirus vectors can be used. A suitable vector for the object of therecombinant DNA is selected.

Furthermore, a transformant can be produced by introducing therecombinant DNA into an appropriate host. The host to be used for theproduction of a transformant is not specifically limited. Microorganismssuch as bacteria, yeasts and filamentous fungi as well as cultured cellsfrom mammals, plants, insects and like can be used. The polypeptide ofthe present invention can be produced in large quantities by culturingthe transformant to produce the polypeptide of the present invention inthe culture.

3. The Method of Producing Glucose, Oligosaccharide or CyclodextrinUsing the Polypeptide of the Present Invention

D-glucose can be released from a polymer of D-glucopyranose likedthrough α-1,4 bonds by using the polypeptide of the present invention.According to the present invention, the degree of polymerization ofglucopyranose in the polymer of D-glucopyranose liked through α-1,4bonds is not specifically limited. Maltose, amylose and starch areincluded. The polymers of D-glucopyranose liked through α-1,4 bondsaccording to the present invention also include a polymer that containsa bond other than the α-1,4 bond (e.g., an α-1,6 bond) or a saccharideother than D-glucose (e.g., fructose) in the molecule. The polypeptideof the present invention having the sequence of SEQ ID NO:1 is highlythermostable. Thus, it can efficiently digest a substrate partially dueto synergistic effects with the change of the conformation and thephysical properties of the substrate upon heating.

Specific reaction conditions are exemplified as follows. If apolypeptide having the amino acid sequence of SEQ ID NO:1 is used,D-glucose can be released by reacting it with substrate in 50 mM sodiumacetate buffer (pH 5.5) at 80° C. Naturally, the optimal reactionconditions may vary depending on the type of the substrate (starch,maltose, etc.).

The polypeptide used for the method for producing glucose of the presentinvention is not limited to an isolated and purified polypeptide. Acrude or partially purified polypeptide may be used as long as it doesnot have harmful influence on the production of glucose. The polypeptideof the present invention may be added to a substrate solution in a freeform. However, the polypeptide is readily recovered after completion ofthe reaction if it is immobilized onto an appropriate carrier andreacted with a substrate.

Furthermore, it is possible to digest starch into D-glucose with highefficiency by using thermostable enzyme(s) such as α-amylase togetherwith the polypeptide of the present invention.

Furthermore, the polypeptide of the present invention can be used toproduce an oligosaccharide and a cyclodextrin.

An oligosaccharide or a cyclodextrin can be produced by reacting starch,amylose or an appropriate oligosaccharide as a substrate underconditions suitable for the polypeptide of the present invention asdescribed above. Naturally, the reaction conditions may be appropriatelyadjusted depending on the type of the product of interest.Oligosaccharides obtained according to the method of the presentinvention are exemplified by maltooligosaccharides from maltose (G2) tomaltooctaose (G8). Cyclodextrins obtained according to the method of thepresent invention are exemplified by α-cyclodextrin, β-cyclodextrin andγ-cyclodextrin.

EXAMPLES

The following examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

Among the procedures described herein, basic procedures includingpreparation of plasmid DNAs and restriction enzyme digestion werecarried out as described in Molecular Cloning: A Laboratory Manual 2nded. (supra). Unless otherwise stated, Escherichia coli JM109 was used asa host for the construction of plasmids using Escherichia coli.Transformed E. coli cells were cultured aerobically at 37° C. using LBmedium (1% Tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0) containing100 μg/ml of ampicillin or LB plate prepared by adding agar atconcentration of 1.5% to LB medium and solidifying the resultingmixture.

Example 1 Isolation of Gene Encoding Polypeptide Having GlucoamylaseActivity

(1) Preparation of Genomic DNA from Pyrococcus furiosus

Pyrococcus furiosus DSM3638 (purchased from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH) was cultured as follows.

2 l of a medium containing 1% Tryptone (Difco Laboratories), 0.5% yeastextract (Difco Laboratories), 1% soluble starch (Nacalai Tesque), 3.5%Jamarine S Solid (Jamarine Laboratory), 0.5% Jamarine S Liquid (JamarineLaboratory), 0.003% MgSO₄, 0.001% NaCl, 0.0001% FeSO₄·7H₂O, 0.0001%CoSO₄, 0.0001% CaCl₂.7H₂O, 0.0001% ZnSO₄, 0.1 ppm CuSO₄·5H₂O, 0.1 ppmKAl(SO₄)₂, 0.1 ppm H₃BO₃, 0.1 ppm Na₂MoO₄·2H₂O, 0.25 ppm NiCl₂·6H₂O wasplaced in a 2-l medium bottle, sterilized at 120° C. for 20 minutes, andbubbled with nitrogen gas to remove dissolved oxygen. Then, theabove-mentioned strain was inoculated into the medium and cultured at95° C. for 16 hours without shaking. After cultivation, cells werecollected by centrifugation.

The resulting cells were then suspended in 4 ml of 50 mM Tris-HCl (pH8.0) containing 25% sucrose. 2 ml of 0.2 M EDTA and 0.8 ml of lysozyme(5 mg/ml) were added to the suspension. The mixture was incubated at 20°C. for 1 hour. 24 ml of SET solution (150 mM NaCl, 1 mM EDTA, 20 mMTris-HCl, pH 8.0), 4 ml of 5% SDS and 400 μl of proteinase K (10 mg/ml)were then added to the mixture. Incubation was further carried out at37° C. for 1 hour. The reaction was terminated by extracting the mixturewith phenol-chloroform. Then, ethanol precipitation was carried out toobtain a genomic DNA.

(2) Preparation of DNA Fragment Containing Gene Encoding PolypeptideHaving Glucoamylase Activity

An oligonucleotide aml-F1 having the nucleotide sequence of SEQ ID NO:3and an oligonucleotide AMY-2N having the nucleotide sequence of SEQ IDNO:4 were synthesized on the basis of the nucleotide sequence ofPyrococcus furiosus genome. A PCR was carried out using these twooligonucleotides as primers and the above-mentioned genornic DNA as atemplate. The PCR was carried out according to the protocol attached toTaKaRa ExTaq (Takara Shuzo) as follows: 30 cycles of 94° C. for 0.5minute, 55° C. for 1 minute and 72° C. for 5 minutes. The reactionmixture was subjected to agarose gel electrophoresis. An amplified DNAfragment of about 3.5-kb was extracted from the gel and purified. Thenucleotide sequence of the about 3.5-kb amplified DNA fragment is shownas SEQ ID NO:5.

(3) Construction of Recombinant Plasmid pSJ3231

The about 3.5-kb amplified DNA fragment obtained in (2) above wasdigested with XbaI and SphI (both from Takara Shuzo), and subjected toagarose gel electrophoresis. A DNA fragment of about 2.3 kb containingthe gene of interest was then extracted and purified. On the other hand,a plasmid vector pUC19 (Takara Shuzo) was digested with Xba I and SphIand dephosphorylated with alkaline phosphatase (Takara Shuzo). The twoDNA fragments were ligated using DNA ligase (Takara Shuzo). The ligationmixture was used to transform Escherichia coli JM109 (Takara Shuzo).Several transformants were selected from the resulting transformants,and the size of each of DNA fragments inserted in plasmid DNAs harboredin the respective transformants was determined. A plasmid having theabout 2.3-kb DNA fragment was purified. The nucleotide sequence of thethus obtained plasmid DNA was determined. Thus, a plasmid pSJ3231 havinga DNA that is derived from the above-mentioned about 3.5-kb DNA fragmentand has a nucleotide sequence of SEQ ID NO:2 was obtained. Escherichiacoli JM109 harboring pSJ3231 was designated as Escherichia coliJM109/pSJ3231.

Example 2 Production of Polypeptide

(1) Expression of Polypeptide

Escherichia coli JM109 harboring pSJ3231 prepared in Example 1 (3) orpUC19 as a vector control was separately inoculated into 5 ml of LBmedium containing 100 μg/ml of ampicillin, and cultured aerobically at37° C. overnight. The culture was inoculated into 20 ml of the samefresh medium at a concentration of 1%, and cultured aerobically at 37°C. When the turbidity reached OD₆₀₀=0.4 to 0.7,isopropyl-β-D-thiogalactopyranoside (IPTG, Takara Shuzo) was added at afinal concentration of 0.2 mM. The cells were further culturedovernight. After cultivation, the cells were collected bycentrifugation, suspended in 0.5 ml of 50 mM sodium acetate buffer (pH5.5), disrupted by sonication and treated at 80° C. for 10 minutes.Supernatants obtained by centrifugation were concentrated by 20 timesusing Ultrafree-MC (Millipore) and used as cell-free extracts fordetermining activities as follows.

5 μl of the cell-free extract was mixed with an equal volume of 2×sample buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.005%Bromophenol Blue). The mixture was applied to SDS-polyacrylamide gelcontaining 0.1% soluble starch in the separation gel and electrophoresedaccording to a conventional method. After electrophoresis, the gel waswashed three times each for 5 minutes in 50 mM sodium acetate buffer (pH5.5) at room temperature and reacted in the same buffer at 80° C. for1.5 hours. After reaction, the gel was briefly washed with water andstained with an iodine solution (an aqueous solution containing 10 mM I₂and 1% KI)

As a result, digestion of starch was not observed for the cell-freeextract prepared from Escherichia coli JM109 harboring pUC19. On theother hand, a clear band resulting from the digestion of starch wasobserved on the gel for the cell-free extract prepared from Escherichiacoli JM109 harboring pSJ3231. The band for the sample obtained with theaddition of 0.2 mM IPTG was more intense than the band for the sampleobtained without the addition of IPTG.

These results show that the polypeptide expressed in Escherichia coliJM109 harboring pSJ3231 has an activity of digesting starch.

(2) Identification of Product Resulting from Hydrolysis of Starch by theAction of Expressed Polypeptide

Before examining, an expressed polypeptide solution was prepared asfollows.

Escherichia coli JM109 harboring pSJ3231 was inoculated into 20 ml of LBmedium containing 100 μg/ml of ampicillin and cultured at 37° C.overnight. The culture was inoculated into 1 liter of the same mediumand cultured at 37° C. until the turbidity reached. OD₆₀₀=0.5. IPTG at afinal concentration of 0.2 mM was added thereto. The cells were furthercultured overnight. The cells were collected by centrifugation,suspended in 40 ml of 50 mM sodium acetate buffer (pH 5.5), sonicatedand treated at 80° C. for 10 minutes. Then, a supernatant was obtainedby centrifugation.

Ammonium sulfate was added to the supernatant to 60% saturation. Themixture was stirred at 4° C. overnight. Precipitates collected bycentrifugation were dissolved in 1 ml of the acetate buffer and dialyzedagainst the same buffer. Precipitates obtained after the dialysis wassuspended in 200 μl of the buffer. The suspension was used as anexpressed polypeptide suspension in the subsequent experiments.

The activity of hydrolyzing starch of the expressed polypeptide wasidentified as follows. Specifically, the expressed polypeptidesuspension was allowed to act on various substrates. Products were thenidentified on thin-layer chromatography.

20 μl of an aqueous solution containing soluble starch (Nacalai Tesque)at a concentration of 5% or a suspension containing amylose (NacalaiTesque) at concentration of 5% in water, 20 μl of the expressedpolypeptide suspension, 10 μl of 500 mM sodium acetate buffer (pH 5.5)and 50 μl of water were mixed together. The mixture was reacted at 80°C. for 17 hours. 2 μl of the reaction mixture was subjected to silicagel thin-layer chromatography using Silica Gel 60F254 (Merck) as a thinlayer plate and ethanol: 1-butanol: water=5:5:3 as a developing solvent.An orcinol-sulfuric acid reagent [prepared by dissolving 400 mg oforcinol (Sigma) in 22.8 ml of sulfuric acid and thereto adding water tomake the total volume up to 200 ml] or a silver nitrate-ammonium reagent(a mixture of equal volumes of 0.1 M silver nitrate and 5 N aqueousammonium) was sprayed to the thin layer plate after development and theplate was heated on a hot plate to observe the spots in order to confirmthe products from the substrates.

As a result, glucose was generated by the action of the expressedpolypeptide using either soluble starch or amylose as a substrate.Glucose was not observed for control experiments in which the expressedpolypeptide or the substrate was used alone.

A reaction was carried out in a similar manner as described above exceptthat a substrate which had been reduced using sodium boron hydride wasused and that the reaction time was 0, 1, 3, 5.5 or 23 hours. Thesubstrate was reduced as follows. 200 mg of starch or amylose wassuspended in 1 ml of water and dissolved by heating. The solution wasdiluted to a volume of 4.5 ml. The resulting solution was cooled on ice.0.5 ml of ice-cold 1.5% sodium boron hydride aqueous solution was slowlyadded thereto. The resulting mixture was reacted at 25° C. for 1 hour.0.1 ml of acetone was added thereto. The mixture was allowed to stand atroom temperature for 20 minutes and then neutralized using 1 N aceticacid. The resulting mixture was used as a substrate solution.

The amount of reducing sugar contained in this reaction mixture wasmeasured according to the Park & Johnson method. Briefly, 10 μl of thereaction mixture which had been appropriately diluted, 40 μl of water,50 μl of a carbonate cyanide solution and 50 μl of 0.05% potassiumferricyanide aqueous solution were mixed together and reacted for 15minutes in boiling water bath. The carbonate cyanide solution wasprepared by dissolving 5.3 g of sodium carbonate and 0.65 g of potassiumcyanide in 1 liter of water. 75 μl of the reaction mixture was mixedwith 125 μl of an iron alum solution. The iron alum solution wasprepared by dissolving 1.5 g of iron alum and 1 g of SDS in 1 liter of0.15 N sulfuric acid. The resulting mixture was allowed to stand at roomtemperature for 15 minutes. Absorbance at 690 nm was then measured. Theamount of reducing end was determined as the amount of correspondingglucose based on a calibration curve prepared using glucose at a knownconcentration. In addition, the amount of glucose in the reactionmixture was measured using Glucose Test Wako (Wako Pure ChemicalIndustries).

As a result, the amounts of reducing sugar and glucose were increasedwith the lapse of reaction time. Thus, it was demonstrated that thepolypeptide of the present invention has a glucoamylase activity.

Example 3 Enzymological Properties of Polypeptide

(1) Preparation of Crude Extract

Escherichia coli JM109 harboring pSJ3231 was inoculated into 20 ml of LBmedium containing 100 μg/ml of ampicillin and cultured at 37° C.overnight. The culture was inoculated into 1 liter of the same mediumand cultured with shaking at 37° C. until the turbidity reachedOD₆₀₀=0.5. IPTG at a final concentration of 0.2 mM was added thereto.The cells were further cultured overnight. The cells were collected bycentrifugation, suspended in 75 ml of 50 mM sodium acetate buffer (pH5.5) and sonicated. A supernatant obtained by centrifuging the sonicatedsuspension was treated at 80° C. for 10 minutes. The resulting insolublesubstances were removed by centrifugation to obtain a supernatant.Ammonium sulfate was added to the supernatant to 60% saturation. Themixture was stirred at 4° C. for 5 hours. Precipitates obtained bycentrifugation were suspended in 1 ml of the sodium acetate buffer anddialyzed overnight against the same buffer. A supernatant obtained bycentrifugation was used as a crude extract in the subsequentexperiments.

(2) Dependency Upon Reaction Temperature

Maltotriose (final concentration of 1%), CaCl₂ (final concentration of 1mM) and the sodium acetate buffer (final concentration of 50 mM) wereadded to 10 μl of the crude extract prepared in Example 3-(1), and thetotal volume was made up to 50 μl. The mixture was reacted at 40, 60,70, 80, 85, 90, 95 or 100° C. for 1 hour. The glucoamylase activity ofthe polypeptide of the present invention was determined by measuring theamount of glucose in the reaction mixture using Glucose Test Wako. As aresult, the polypeptide of the present invention exhibited the maximalglucoamylase activity at 85 to 90° C.

The results are shown in FIG. 1. FIG. 1 illustrates the relationshipbetween the glucoamylase activity of the polypeptide of the presentinvention and the reaction temperature. The horizontal axis representsthe reaction temperature (° C.) and the vertical axis represents theglucoamylase activity (relative value, %).

(3) Dependency Upon Reaction pH

Maltotriose (final concentration of 1%) and a buffer (sodium acetate,MES-NaOH, sodium phosphate, Tris-HCl or glycine-NaOH; finalconcentration of 50 mM) were added to 10 μl of the crude extractprepared in Example 3-(1), and the total volume was made up to 50 μl.The mixture was reacted at 80 C for 1 hour. The pH of each buffer wasadjusted at 80 C. The glucoamylase activity of the polypeptide of thepresent invention was determined by measuring the amount of glucose inthe reaction mixture using Glucose Test Wako. As a result, thepolypeptide of the present invention exhibited maximum glucoamylaseactivity at 5 to 6.

The results are shown in FIG. 2. FIG. 2 illustrates the relationshipbetween the glucoamylase activity of the polypeptide of the presentinvention and the reaction pH. The horizontal axis represents thereaction pH and the vertical axis represents the glucoamylase activity(relative value, %). In FIG. 2, open circles (∘), closed circles (●),open squares (□), closed squares (▪) and open triangles (Δ) representthe results for sodium acetate buffers, MES-NaOH buffers, sodiumphosphate buffers, Tris-HCl buffers and glycine-NaOH buffers,respectively.

(4) Heat Stability

An equal volume of 50 mM sodium acetate buffer (pH 5.5), the same buffercontaining 2 mM EDTA or the same buffer containing 2 mM CaCl₂ was addedto the crude extract prepared in Example 3-(1). The mixture was heatedat 80 or 95° C. for 1, 5 or 24 hours. Maltotriose (final concentrationof 1%), the sodium acetate buffer (final concentration of 50 mM) andCaCl₂ (final concentration of 1 mM) were added to 30 μl of the heatedmixture, and the total volume was made up to 50 μl. The mixture wasreacted at 80° C. for 1 hour. The remaining activity relative to thecrude extract without the heat treatment was determined by measuring theamount of glucose in the reaction mixture using Glucose Test Wako. As aresult, almost no inactivation was observed when the crude extract washeated at 80° C. in the absence of EDTA for 24 hours. When the crudeextract was heated at 95° C., most of the activity was lost afterheating for 1 hour without the addition of CaCl₂. On the other hand,with the addition of CaCl₂ at a concentration of 1 mM, no Inactivationwas observed after heating for 5 hours and a slight remaining activitywas observed after heating for 24 hours.

The results are shown in FIGS. 3 and 4. FIGS. 3 and 4 illustrate theremaining glucoamylase activity of the polypeptide of the presentinvention upon heating at 80° C. and 95° C., respectively. Thehorizontal axes represent the heat treatment time (hour) and thevertical axes represent the remaining glucoamylase activity (%). InFIGS. 3 and 4, open circles (∘), closed circles (●) and open squares (□)represent the results for the heat treatment without the addition ofEDTA or CaCl₂, the heat treatment with the addition of 1 mM EDTA and theheat treatment with the addition of 1 mM CaCl₂, respectively.

Additionally, when a remaining activity was determined after heating at95° C. for 24 hours in the presence of 10 mM CaCl₂ in a similar manneras described above, almost 100% of the activity was retained.

(5) pH Stability

20 μl of 100 mM buffer (sodium acetate, MES-NaOH, sodium phosphate,Tris-HCl or glycine-NaOH) was added to 20 μl of the crude extractprepared in Example 3-(1). The mixture was heated at 80° C. for 10minutes. Maltotriose (final concentration of 1%) and the sodium acetatebuffer (final concentration of 50 mM) were added to 30 μl of the heatedmixture, and the total volume was made up to 50 μl. The mixture wasreacted at 80° C. for 1 hour. The remaining activity relative to thecrude extract without the heat treatment was determined by measuring theamount of glucose in the reaction mixture using Glucose Test Wako. As aresult, 50% or more of the activity remained after heating at pH 5 to 9.

(6) Effects of Metal Ions

Maltotriose (final concentration of 1%), the sodium acetate buffer (pH5.5, final concentration of 50 mM) and CoCl₂, CaCl₂, CuSO₄, FeCl₃,ZnCl₂, MgCl₂ or EDTA (final concentration of 0, 0.5, 1, 2 or 10 mM) wereadded to 10 μl of the crude extract prepared in Example 3-(1), and thetotal volume was made up to 50 μl. The mixture was reacted at 80° C. for1 hour. The glucoamylase activity of the polypeptide of the presentinvention was determined by measuring the amount of glucose in thereaction mixture using Glucose Test Wako. As a result, the glucoamylaseactivity of the polypeptide of the present invention was notsignificantly influenced by CoCl₂, CaCl₂, FeCl₃ or MgCl₂ at aconcentration of 0.5, 1, 2 or 10 mM. The activity was completelyinhibited by CuSO₄ or ZnCl₂ at a concentration of 2 mM, or EDTA at aconcentration of 0.5 mM.

The results are shown in FIG. 5. FIG. 5 illustrates the relationshipbetween the concentration of a metal ion or EDTA and the glucoamylaseactivity of the polypcptide of the present invention. The horizontalaxis represents the concentration of the metal ion or EDTA added and thevertical axis represents the glucoamylase activity (relative value, %).In FIG. 5, open circles (∘), closed circles (●), open squares (□),closed squares (▪), open triangles (Δ), closed triangles (▴) andasterisks (*) represent the results for the addition of CoCl₂, CaCl₂,CuSO₄, FeCl₃, ZnCl₂, MgCl₂ and EDTA, respectively.

Example 4 Properties of Mutant Polypeptides

(1) Construction of Recombinant Plasmid pET21amyCΔS

An oligonucleotide PX253-01 having a nucleotide sequence of SEQ ID NO:8and an oligonucleotide R4 having a nucleotide sequence of SEQ ID NO:9were synthesized. A PCR was carried out using these two oligonucleotidesas primers and pSJ3231 as a template. The PCR was carried out accordingto the protocol attached to TaKaRa ExTaq as follows: 30 cycles of 94° C.for 0.5 minute, 55° C. for 0.5 minute and 72° C. for 2 minutes. A DNAwas recovered from the reaction mixture by ethanol precipitation,digested with NcoI and EcoRI (both from Takara Shuzo) and then subjectedto agarose gel electrophoresis. An about 1.0-kb DNA fragment wasextracted and purified from the gel. The DNA fragment was ligated topET21d (Novagen) digested with NcoI and EcoRI using T4 DNA ligase. Theligation mixture was used to transform Escherichia coli JM109. Plasmidswere prepared from the resulting transformants to obtain pRH03. Theabout 1.0-kb DNA fragment was inserted into pRH03. When the nucleotidesequence was determined, it was shown that the fragment contained asequence CCATGG recognized by NcoI followed by a sequence from C atposition 2 to C at position 1048 in the nucleotide sequence of SEQ IDNO:7.

The about 3.5-kb amplified DNA fragment prepared in Example 1-(2) wasdigested with AflII and SacI (both from Takara Shuzo) and subjected toagarose gel electrophoresis. An about 1.8-kb DNA fragment was extractedand purified from the gel. This DNA fragment was ligated to pRH03digested with AflII and SacI using T4 DNA ligase. The ligation mixturewas used to transform Escherichia coli JM109. Plasmids were preparedfrom the resulting transformants to obtain pET21amyCΔS . When thenucleotide sequence of pET21amyCΔS was determined, it was shown that theplasmid has a polynucleotide having a nucleotide sequence of SEQ IDNO:7, and the nucleotide sequence encodes a polypeptide having an aminoacid sequence of SEQ ID NO:6.

(2) Construction of Expression Plasmids for Mutant Polypeptides

The following oligonucleotides were synthesized: an oligonucleotideF206S-F having a nucleotide sequence of SEQ ID NO:10; an oligonucleotideF206S-R having a nucleotide sequence of SEQ ID NO:11; an oligonucleotideP142I-F having a nucleotide sequence of SEQ ID NO:12; an oligonucleotideP142I-R having a nucleotide sequence of SEQ ID NO:13; an oligonucleotideL337V-F having a nucleotide sequence of SEQ ID NO:14; an oligonucleotideL337V-R having a nucleotide sequence of SEQ ID NO: 15; anoligonucleotide F2 having a nucleotide sequence of SEQ ID NO:16; anoligonucleotide F3 having a nucleotide sequence of SEQ ID NO:17; anoligonucleotide AN2-R1F2 having a nucleotide sequence of SEQ ID NO:18;an oligonucleotide R5 having a nucleotide sequence of SEQ ID NO:19; andan oligonucleotide R6 having a nucleotide sequence of SEQ ID NO:20.

PCRs were carried out using pSJ3231 as a template and theoligonucleotides as shown in Table 1 as primers. The PCRs were carriedout according to the protocol attached to TaKaRa ExTaq as follows: 20cycles of 94° C. for 0.5 minute, 55° C. for 0.5 minute and 72° C. for 1minute. The PCR reaction mixtures were subjected to agarose gelelectrophoresis. DNA fragments each having the chain length as shown inTable 1 were extracted and purified from the gel.

TABLE 1 Reaction FS-F FS-R PI-F PI-R LV-F LV-R Primer 1 F206 F206 P142P142 L337 L337 S-F S-R I-F I-R V-F V-R Primer 2 R5 F2 R6 AN2- R4 F3 R1F2Chain length (bp) about about about about about about 400 260 290 260300 350

Amplified DNA fragments purified as described above were used for PCRsas follows. PCRs were further carried out using the combinations of thepurified DNA fragments as templates and the primers as shown in Table 2.“Template 1” and “template 2” indicated in Table 2 correspond to“reaction” in Table 1, indicating that the DNA fragments from thereactions shown in Table 1 were used in the reactions shown in Table 2as templates. The PCRs were carried out according to the protocolattached to TaKaRa ExTaq as follows: 10 cycles of 94° C. for 0.5 minute,55° C. for 0.5 minute and 72° C. for 1 minute. The PCR reaction mixtureswere subjected to agarose gel electrophoresis. DNA fragments each havingthe chain length as shown in Table 2 were extracted and purified fromthe gel.

TABLE 2 Reaction FS PI LV Template 1 FS-F PI-F LV-F Template 2 FS-R PI-RLV-R Primer 1 F2 AN2-R1F2 F3 Primer 2 R5 R6 R4 Chain length (bp) about650 about 530 about 640

The DNA fragment from the reaction FS in Table 2 was digested with AflIIand NdeI (Takara Shuzo) and subjected to agarose gel electrophoresis. Anabout 320-bp DNA fragment was extracted and purified from the gel. Thisfragment was ligated to pET21amyCΔS digested with AflII and NdeI usingT4 DNA ligase. The ligation mixture was used to transform Escherichiacoli JM109. Plasmids were prepared from the resulting transformants toobtain pamyCΔS-F206S. When the nucleotide sequence of pamyCΔS-F206S DNAwas determined, it was shown that the plasmid encodes a polypeptidehaving an amino acid sequence of SEQ ID NO:6 in which Phe at position187 (corresponding to position 206 in the amino acid sequence of SEQ IDNO:1) is replaced by Ser. This mutant polypeptide was designated asF206S.

The DNA fragment from the reaction PI in Table 2 was digested with BalI(Takara Shuzo) and AflII and subjected to agarose gel electrophoresis.An about 280-bp DNA fragment was extracted and purified from the gel.This fragment was ligated to pET21amyCΔS digested with BalI and AflIIusing T4 DNA ligase. The ligation mixture was used to transformEscherichia coli JM109. Plasmids were prepared from the resultingtransformants to obtain pamyCΔS-P142I. When the nucleotide sequence ofpamyCΔS-P142I DNA was determined, it was shown that the plasmid encodesa polypeptide having an amino acid sequence of SEQ ID NO:6 in which Proat position 123 (corresponding to position 142 in the amino acidsequence of SEQ ID NO:1) is replaced by Ile. This mutant polypeptide wasdesignated as P142I.

The DNA fragment from the reaction LV in Table 2 was digested with NdeIand EcoRI and subjected to agarose gel electrophoresis. An about 170-bpDNA fragment was extracted and purified from the gel. This fragment wasligated to pET21amyCΔS digested with NdeI and EcoRI using T4 DNA ligase.The ligation mixture was used to transform Escherichia coli JM109.Plasmids were prepared from the resulting transformants to obtainpamyCΔS-L337V. When the nucleotide sequence of pamyCΔS-L337V DNA wasdetermined, it was shown that the plasmid encodes a polypeptide havingan amino acid sequence of SEQ ID NO:6 in which Leu at position 318(corresponding to position 337 in the amino acid sequence of SEQ IDNO:1) is replaced by Val. This mutant polypeptide was designated asL337V.

(3) Production of Wild Type and Mutant Polypeptides

Escherichia coli BL21(DE3) (Novagen) was transformed with pET21amyCΔS,pamyCΔS-F206S, pamyCΔS-P142I or pamyCΔS-L337V. Each of the resultingtransformants was inoculated into 20 ml of LB medium containing 100μg/ml of ampicillin and cultured aerobically at 37° C. overnight. Aftercultivation, the cells were collected by centrifugation, suspended in0.8 ml of 50 mM sodium acetate buffer (pH 5.5), disrupted by sonicationand treated at 80° C. for 30 minutes. A supernatant obtained bycentrifugation was used as a crude extract. Hereinafter, crude extractsfrom Escherichia coli BL21(DE3) transformed with pET21amyCΔS,pamyCΔS-F206S, pamyCΔS-P142I and pamyCΔS-L337V are referred to as acrude extract WT, a crude extract F206S, a crude extract P142I and acrude extract L337V, respectively.

(4) Digestion of Maltooligosaccharides with Wild Type and MutantPolypeptides

Maltotriose (final concentration of 1%), sodium acetate buffer (pH 5.5,final concentration of 50 mM) and CaCl₂ (final concentration of 1 mM)were added to 5 μl of one of the crude extracts obtained in Example4-(3), and the total volume was made up to 50 μl. The mixture wasreacted at 80° C. for 1 hour. The glucoamylase activity of thepolypeptide of the present invention contained in the crude extract wasdetermined by measuring the amount of glucose in the reaction mixtureusing Glucose Test Wako. As a result, provided that the glucoamylaseactivity for the crude extract WT was defined as 1, the glucoamylaseactivities for the crude extract F206S, the crude extract P1421 and thecrude extract L337V were determined to be 2.08, 0.55, and 0.30,respectively.

Similar reactions were carried out using maltopentaose in place ofmaltotriose. As a result, provided that the glucoamylase activity forthe crude extract WT was defined as 1, the glucoamylase activities forthe crude extract F206S, the crude extract P142I and the crude extractL337V were determined to be 1.92, 0.53, and 0.37, respectively.

65 μl of acetonitrile was added to and mixed with 35 μl of the reactionmixture obtained as described above using maltopentaose as a substrate.The mixture was centrifuged to remove insoluble substances. Thecomposition of oligosaccharides contained in the sample was analyzed bynormal phase HPLC using Palpak Type S (Takara Shuzo). 65% acetonitrileaqueous solution was used for the mobile phase. The flow rate was 1ml/minute and the column temperature was 40° C. Detection was carriedout using a differential refractive index detector Shodex RI-71 (ShowaDenko). 65 μl of acetonitrile was added to 35 μl of each of aqueoussolution containing glucose (Nacalai Tesque), maltose, maltotriose,maltopentaose or maltoheptaose (Seikagaku Corporation) at aconcentration of 0.1%. Supernatants obtained by centrifuging themixtures were used as standards. The retention times for the standardswere as follows: 5.6 minutes (glucose), 7.3 minutes (maltose), 9.7minutes (maitotriose), 17.8 minutes (maltopentaose) arid 30.7 minutes(maltoheptaose). As a result, it was shown that maltooligosaccharideshaving degrees of polymerization ranging from 1 to 8 were generated whenthe respective crude extracts were allowed to act on maltopentaose. Theresults are shown in FIG. 6. FIG. 6 illustrates the composition ofproducts obtained by allowing the respective crude extracts to act onmaltopentaose. The horizontal axis represents the products (G1: glucose;G2: maltose; G3: maltotriose; G4: maltotetraose; G6: maltohexaose; G7:maltoheptaose; and G8: maltooctaose) and the vertical axis representsthe concentration of the product (as the concentration of correspondingglucose; μM) in the reaction mixture. In FIG. 6, open circles (∘),closed circles (●), open squares (□) and closed squares (▪) representthe results for the crude extract WT, the crude extract F206S, the crudeextract P142I and the crude extract L337V, respectively. The amounts ofgenerated glucose determined using HPLC as described above are notconsistent with the results obtained using Glucose Test Wako. It isconsidered that this is because a peak for the solvent appeared at theposition at which glucose was eluted in HPLC, and thus the measurementfor glucose included an error.

(5) Digestion of Starch with Wild Type and Mutant Polypeptides

A hyperthermostable α-amylase preparation was prepared according to amethod as described in Example 3 of JP-A 7-143880 entitled“Hyperthermostable α-amylase gene.” The following volumes of the crudeextract F206S and the hyperthermostable α-amylase preparation were used:40 μl and 0 μl (reaction 1); 30 μl and 10 μl (reaction 2); 20 μl and 20μl (reaction 3); 10 μl and 30 μl (reaction 4); and 0 μl and 40 μl(reaction 5). Soluble starch (Nacalai Tesque; final concentration of2%), sodium acetate buffer (pH 5.5; final concentration of 50 mM) andCaCl₂ (final concentration of 1 mM) were added thereto, and the totalvolume was made up to 200 μl. The mixture was reacted at 80° C.overnight. The oligosaccharides in the reaction mixtures were analyzedby normal HPLC as described in Example 4-(4). As compared with theresults obtained by using the hyperthermostable α-amylase preparation orthe crude extract F206S alone, the sum of the concentrations (as theconcentrations of corresponding glucose) of glucose, maltose andmaltotriose, which can be fermented by a yeast commonly used for alcoholfermentation, was greater when these enzymes were used in combination.The results are shown in FIG. 7. FIG. 7 illustrates the composition ofproducts obtained by allowing the respective crude extracts to act onsoluble starch. The horizontal axis represents the products (G5:maltopentaose; others are described above for FIG. 6) and the verticalaxis represents the concentration of the product in the reaction mixture(as the concentration of corresponding glucose; μM). In FIG. 7, opencircles (∘), open triangles (Δ), open squares (□), closed triangles (▴)and closed circles (●) represent the results for the reaction 1, thereaction 2, the reaction 3, the reaction 4 and the reaction 5,respectively.

(6) Activity of Synthesizing Cyclodextrin of the Polypeptide of thePresent Invention

2 units of glucoamylase from Aspergillus niveus (Seikagaku Corporation)were added to 50 μl of the reaction mixture of the reaction 1 in Example4-(5). The mixture was reacted at 37° C. for 3 hours. When the reactionmixture was analyzed by normal phase HPLC as described in Example 4-(4),peaks having the same retention times as those observed forα-cyclodextrin (CD), β-CD and γ-CD (Seikagaku Corporation) wereobserved. The retention times for α-CD, β-CD and γ-CD were 12.3 minutes,15.2 minutes and 19.7 minutes, respectively. The fractions correspondingto these peaks were collected and subjected to mass spectrometry in thepositive ion mode using a triple stage quadrupole mass spectrometer API300 (Perkin-Elmer Sciex) As a result, the samples from the respectivepeaks resulted in the same mass spectra as those of α-CD, β-CD and γ-CD.Thus, it was shown that the polypeptide of the present inventioncontained in the crude extract F206S has an activity of synthesizing CD.

(7) Construction of Expression Plasmids for Double-mutant Polypeptides

pamyCΔS-F206S was digested with PstI (Takara Shuzo) and AflII, andsubjected to agarose gel electrophoresis. An about 3.1-kb DNA fragmentwas extracted and purified from the gel to obtain a DNA 1. pamyCΔS-P142Iwas digested with PstI and AflII, and subjected to agarose gelelectrophoresis. An about 4.6-kb DNA fragment was extracted and purifiedfrom the gel to obtain a DNA 2. pamyCΔS-F206S was digested with PstI andNdeI, and subjected to agarose gel electrophoresis. An about 4.9-kb DNAfragment was extracted and purified from the gel to obtain a DNA 3.pamyCΔS-L337V was digested with PstI and NdeI, and subjected to agarosegel electrophoresis. An about 2.8-kb DNA fragment was extracted andpurified from the gel to obtain a DNA 4.

The DNA 1 and the DNA 2 were ligated each other using T4 DNA ligase. Theligation mixture was used to transform Escherichia coli HB101 (TakaraShuzo). A plasmid pamyCΔS-F206S/P142I was obtained from a transformant.When the nucleotide sequence of the pamyCΔS-F206S/P142I DNA wasdetermined, it was shown that the plasmid encodes a polypeptide havingan amino acid sequence of SEQ ID NO:6 in which Phe at position 187 isreplaced by Ser and Pro at position 123 is replaced by Ile. This mutantpolypeptide was designated as FS/PI.

The DNA 3 and the DNA 4 were ligated each other using T4 DNA ligase. Theligation mixture was used to transform Escherichia coli HB101. A plasmidpamyCΔS-F206S/L337V was obtained from a transformant. When thenucleotide sequence of the pamyCΔS-F206S/L337V DNA was determined, itwas shown that the plasmid encodes a polypeptide having an amino acidsequence of SEQ ID NO:6 in which Phe at position 187 is replaced by Serand Leu at position 318 is replaced by Val. This mutant polypeptide wasdesignated as FS/LV.

(8) Properties of Double-mutant Polypeptides

Escherichia coli BL21(DE3) was transformed with pamyCΔS-F206S/P142I orpamyCΔS-F206S/L337V. A crude extract FS/PI and a crude extract FS/LVwere obtained using the transformants as described in Example 4-(3).

Soluble starch (final concentration of 2%), sodium acetate buffer (pH5.5; final concentration of 50 mM) and CaCl₂ (final concentration of 1mM) were added to 20 μl of the crude extract WT, the crude extract F206S(both prepared in Example 4-(3)), the crude extract FS/PI or the crudeextract FS/LV, and the total volume was made up to 100 μl. The mixturewas reacted at 80° C. overnight. The oligosaccharides in the reactionmixtures were analyzed by normal phase HPLC as described in Example4-(4). As a result, provided that the sum of the concentrations (as theconcentrations of corresponding glucose) of glucose, maltose andmaltotriose for the crude extract WT was defined as 1, the values forthe crude extract F206S, the crude extract FS/PI and the crude extractFS/LV were determined to be 2.22, 2.22, and 2.79, respectively.

Regarding the composition of produced CDs, the amount of γ-CD was abouthalf the amount of β-CD when the crude extract WT was used. The amountsof γ-CD and β-CD were almost equivalent when the crude extract F206S orthe crude extract FS/LV was used. The amount of γ-CD was about 1.7-foldmore than that of β-CD when the crude extract FS/PI was used. Theresults are shown in Table 3.

TABLE 3 Produced amount (μM) Crude extract β-CD γ-CD WT 220 103 F206S292 294 FS/PI 227 386 FS/LV 379 363Industrial Applicability

The present invention provides a polypeptide having a glucoamylaseactivity. The polypeptide of the present invention is highlythermostable and can efficiently digest starch. Furthermore, it ispossible to efficiently produce glucose from starch by using thepolypeptide of the present invention in combination with an α-amylasefrom a hyperthermophile, facilitating the utilization of biomass.

Sequence Listing Free Text

SEQ ID NO:3: PCR primer am1-F1 for amplifying a gene encoding apolypeptide having a glucoamylase activity from Pyrococcus furiosus.

SEQ ID NO:4 PCR primer AMY-2N for amplifying a gene encoding apolypeptide having a glucoamylase activity from Pyrococcus furiosus.

SEQ ID NO:8: PCR primer PX253-01.

SEQ ID NO:9: PCR primer R4.

SEQ ID NO:10: PCR primer 206S-F.

SEQ ID NO:11: PCR primer F206S-R.

SEQ ID NO:12: PCR primer P142I-F.

SEQ ID NO:13: PCR primer P142I-R.

SEQ ID NO:14: PCR primer L337V-F.

SEQ ID NO:15: PCR primer L337V-R.

SEQ ID NO:16: PCR primer F2.

SEQ ID NO:17: PCR primer F3.

SEQ ID NO:18: PCR primerAN2-R1F2.

SEQ ID NO:19: PCR primer R5.

SEQ ID NO:20: PCR primer R6.

1. An isolated nucleic acid encoding a polypeptide having a thermostableglucoamylase activity selected from the group consisting of: (a) anucleic acid encoding a polypeptide having the amino acid sequence ofSEQ ID NO:6 or an amino acid sequence that differs from SEQ ID NO:6 byone to up to three amino acid substitution(s) selected from the groupconsisting of: i. substitution of Ser for Phe at position 187; ii.substitution of Ile for Pro at position 123; iii. substitution of Valfor Leu at position 318; (b) a nucleic acid having the nucleotidesequence of SEQ ID NO:7.
 2. A recombinant DNA containing the nucleicacid defined by claim
 1. 3. A host cell transformed with the recombinantDNA defined by claim
 2. 4. A method for producing a polypeptide having athermostable glucoamylase activity, the method comprising culturing thehost cell defined by claim 3 and collecting a polypeptide having athermostable glucoamylase activity from the culture.